Keratan sulfate is a glycosaminoglycan having N-acetyllactosamine as the basic structure which has O-sulfated hydroxyl group at C-6 position of the N-acetylglucosamine residue. Especially, high-sulfated keratan sulfate which further has a sulfated hydroxyl group beside that at C-6 position of N-acetylglucosamine residue in the constitutional disaccharide unit is known to be contained in cartilaginous fishes such as sharks, and cartilage, bone and cornea of mammals such as whale and bovines. As a method for producing a keratan sulfate oligosaccharide which is the degradation product of the keratan sulfate, a method which comprises allowing a keratan sulfate degrading enzyme (keratanase II; Japanese Patent Application Laid-open No. 2-57182) derived from the microorganism belonging to the genus Bacillus to act on keratan sulfate has been reported.
Further, there is a report (Biochemistry, 33, 4836-4846 (1994)) which analyzes 25 kinds of the oligosaccharide fractions obtained by the fractionation of degradation products after degrading keratan sulfate derived from bovine cartilage with keratanase II and infers the structures of the tetrasulfated N-acetyllactosamine tetrasaccharide represented by the following formula (I) (hereinafter also referred to as "keratan sulfate tetrasaccharide (I)"), the trisulfated N-acetyllactosamine pentasaccharide represented by the following formula (II) (hereinafter also referred to as "keratan sulfate pentasaccharide (II)"), the disulfated N-acetyllactosamine disaccharide represented by the following formula (III) (hereinafter also referred to as "keratan sulfate disaccharide (III)"), and the like. EQU Gal(6S).beta.1-4GlcNAc(6S).beta.1-3Gal(6S).beta.1-4GlcNAc(6S)(I) EQU NeuAc.about.Gal.beta.1-4GlcNAc(6S).beta.1-3Gal(6S).beta.1-4GlcNAc(6S)(II) EQU Gal(6S).beta.1-4GlcNAc(6S) (III)
(In the foregoing formulae, Gal represents a galactose, GlcN represents a glucosamine, Neu represents a neuraminic acid, Ac represents an acetyl group, and 6S represents a 6-O-sulfate ester. And ".about." represents an .alpha.2,3 bond or .alpha.2,6 bond.)
Up to now, however, no reports have appeared concerning efficient mass production of pure keratan sulfate oligosaccharide, especially the keratan sulfate tetrasaccharide (I), the keratan sulfate pentasaccharide (II), and the keratan sulfate disaccharide (III), without contamination of impurities (for example, endotoxin, nucleic acid, protein, protease, other glycosaminoglycans except for keratan sulfate oligosaccharide, and the like). The contamination of such impurities is liable to be a serious defect especially in the case where the keratan sulfate oligosaccharide is used as pharmaceuticals. Furthermore, the pharmacological functions (especially anti-inflammatory function, antiallergic function, immunomodulating function, cell differentiation inducing function, and apoptosis inducing function) of the keratan sulfate oligosaccharide are not known at all.